How to elute this protein?! - (May/07/2009 )

Hi there,

I'm currently doing IPs for a flag fusion protein I'm working on. Western blot showed that the protein is indeed bound to the resin (M2 flag antibody agarose beads from Sigma) since no protein can be detected in the supernatant post-IP.

The problem I have is that I can't elute "by soft means" my protein. I've tried eluting by competition (1mg/ml of Flag peptide for an hour at 4 C). I ended with barly enough protein to see a shadow of a band in WB.

Then I tried with Glycine at pH 3.5 for 5 minutes at RT. Same results : barely enough protein to see in WB.

Now the problem I have, is that I want to elute my protein and identify by silver stainint/mass spec proteins bound to it. So eluting in 2x laemli buffer surely works, by my bands would be contaminated by the huge amount of Ig that come off the beads.

Now I have two questions :

1- Do you guys know any other elution methods than those I mentioned?

2- When I tried and failed to elute with Glycine, I had kept the beads at -20. Now I tried to elute with 5mg/ml Flag peptide at RT for an hour, but it still didn't work. But I realized when I added laemli buffer to load on a gel that the buffer turned yellow, whitch means that the pH was still very acidic. So, is it possible that the elution with 5mg/ml Flag peptide did not work because the competition for the antibody was abolished by the acidic pH?

Thanks in advance.

Madrius

-madrius1-

That is really odd. Do you get your flag peptide right? A general method is elution by pH disruption, with 100 mM Glycine pH 3.0. The eluted protein should be immediately neutralized with 1mM Tris-HCl, pH 8.0.

James
Understanding Life One Protein At a Time...
www.lifetein.com
A peptide synthesis company.

-James Chou-

Here I am, almost 3 months later, and still nothing improved.

I now perform my IPs with IgG sepharose fast flow, since my protein has two IgG binding domain tags in it. I've tried many things, the last to date being to crosslink proteins with 1% formaldehyde, and then try to wash extensively, after IP, to get a clean eluate. Now, the IP didn't work..

I plan to try with 0,5% formaldehyde, as it has been shown that high % of formaldehyde results in protein precipitation.

Do you guys have any cues to help me with this project? Any basic knowledge I might not have, any dark secret tips about protein purification to identify its binding partners by mass spec?

-madrius1-

To back up a bit.....

For your flag elution, do you have a 1x or a 3x flag-tagged protein? 3x flag tagged proteins are very poorly eluted with flag peptide. If you have a 1x flag-tagged protein, are you eluting with a 1x or a 3x flag peptide?

An alternative method of elution is 2M MgCl2.

It is possible that when you tried to elute your protein with the acidic glycine your protein crashed out of the solution. (and you did neutralize the solution after the acidic glycine treatment!) Not all proteins are happy about having the pH go up and down and will denature out of solution.

A simple way to test if your samples are not eluting or are crashing out of solution is to run the supernatant after the elution step. After you remove the beads, wash them a few times and then boil them in loading buffer.

-miBunny-

A simple way to test if your samples are not eluting or are crashing out of solution is to run the supernatant after the elution step. After you remove the beads, wash them a few times and then boil them in loading buffer.

I already did that. And the protein just didn't elute with glycine. 2X laemli buffer elution gave me a really strong signal in WB.

Now, as soon as I get my IP to work with formaldehyde, i'll try eluting with 2M MgCl2. Last attempt, I tried to elute with 1M NaCl, but got nothing, since the IP had not worked.

Thanks a lot!

Any other input on troubleshooting IP after formaldehyde crosslink?

-madrius1-

madrius1 on Jul 30 2009, 04:18 PM said:

A simple way to test if your samples are not eluting or are crashing out of solution is to run the supernatant after the elution step. After you remove the beads, wash them a few times and then boil them in loading buffer.

I already did that. And the protein just didn't elute with glycine. 2X laemli buffer elution gave me a really strong signal in WB.

Now, as soon as I get my IP to work with formaldehyde, i'll try eluting with 2M MgCl2. Last attempt, I tried to elute with 1M NaCl, but got nothing, since the IP had not worked.

Thanks a lot!

Any other input on troubleshooting IP after formaldehyde crosslink?

A very recent paper "Utilization of Arg-elution method for FLAG-tag based chromatography" suggests 0-5 - 1 M arginine pH 3.5 - 4.4. It is available online (doi:10.1016/j.pep.2009.03.012). Will be in journal Oct 2009. Protein Expression and Purification 67, 148-155